The fundamental role of mitochondria in energy metabolism and respiration was investigated in stem cells from most of the sources (embryonic, adult, cord blood, and induced pluripotent stem cells (IPSC)). However, the particular mitochondrial function in cancer stem cells is still under investigation. Self-renewal capacity and pluripotency are the essential features of stem cells. Several tissue stem cells (for example, long-term hematopoietic stem cells (LT-HSCs) in the bone marrow niche) maintain a quiescent state, as this is essential for preserving their self-renewal capacity. Some types of stem cells heavily rely on anaerobic glycolysis to maintain such a quiescent state and are more sensitive to oxidative stress. In hypoxic conditions (such as those found in the stem cell niche), the transcription factor hypoxia-inducible factor 1α (HIF 1α) promotes glycolysis as it induces the expression of pyruvate dehydrogenase kinases (PDKs), which prevent pyruvate from entering the tricarboxylic acid cycle, thus blocking mitochondrial respiration. The CSC mitochondria have been shown recently to be an important target for cancer treatment, but clinical significance of CSCs and their mitochondria properties remain unclear. Mitochondrial metabolism is altered in cancer cells because of their reliance on glycolytic intermediates, which are normally destined for oxidative phosphorylation. Therefore, inhibiting cancer-specific modifications in mitochondrial metabolism, increasing reactive oxygen species production, or stimulating mitochondrial permeabilization transition could be promising new therapeutic strategies to activate cell death in CSCs as well, as in general cancer cells. Mitochondria-targeted agents are considerably more effective compared to other agents in triggering apoptosis of CSCs, as well as general cancer cells, via mitochondrial dysfunction. This chapter will analyze mitochondrial function in general and in cancer stem cells, and its potential as a therapeutic target to induce cell death in CSCs. Some authors believe that combined treatment with mitochondria-targeted drugs could be a promising strategy for the treatment of relapsed and refractory cancer, which is based upon CSCs.